ABSTRACT Stem cell transplantation in the form of bone marrow, peripheral blood or cord blood-derived cells is commonly performed for reconstitution of the hematopoietic system of patients suffering from cancers and non-malignant diseases. Current practice for cryopreservation of hematopoietic and mesenchymal stem cells is to employ the cell permeating cryoprotectant dimethylsulfoxide, often with an additional non-permeating cryoprotectant, and long-term storage in vapor phase nitrogen (?-135C). The ultimate objective of this proposal is to develop a cryopreservation method that avoids concerns associated with dimethylsulfoxide and allows cells to be stored long-term at or below -80C for years. We have developed prototypic freezing protocols based upon a nanoparticle delivery strategy to load stem cells with a disaccharide trehalose. This technology avoids use of the commonly employed cell permeating cryoprotectant, dimethylsulfoxide, which has patient cytotoxicity concerns, by replacing it with trehalose. Trehalose accumulation is a mechanism used in nature by organisms to survive freezing conditions. In this proposal we plan to evaluate nanoparticle delivered trehalose (nTre) using methods developed by our collaborator, Prof. He. He has demonstrated preservation of human adipose- derived stem cells using nTre without compromising either the stemness or multilineage differentiation potential of the cells. This proposal is to further test the nTre DMSO-free cryopreservation of bone marrow-derived stem cells with the following two specific aims: In Aim 1 we will optimize bone marrow stem cell cryopreservation with nTre. Bone marrow-derived hematopoietic and mesenchymal stem cells will be evaluated with appropriate controls. Assays for stemness and differentiation to evaluate the post-thawing outcomes will be performed on the cryopreserved cells versus fresh controls. In Aim 2 we will validate the optimized cryopreservation method for long-term storage for up to 3 months at both -80C and ?-135C for both stem cell types. The proposed studies will be considered successful if the nTre preserves viability and function, differentiation potential, of >80% of fresh control bone marrow stem cells. In a future Phase II SBIR proposal we will verify the hematopoietic reconstitution potential of human disaccharide-preserved stem cells by transplantation in a humanized mouse model. The reconstitution potential of the cryopreserved cells will also be evaluated versus controls by analysis of human cell markers and secondary transplantation.